"non-natural peptides targeting the scaffold activity of PI3kγ and therapeutic uses thereof"

Abstract

A non-natural peptide having the ability of inhibiting the A-kinase anchoring function of PI3Kγ comprising an amino acid sequence as set forth in SEQ ID No.: 1, wherein each amino acid is a D-amino acid, therapeutic uses and composition thereof.

Description

[0001] Non-natural peptides targeting the scaffold activity of

[0002] PI3Ky and therapeutic uses thereof"

[0003] FIELD OF THE INVENTION

[0004] The present description relates to non-natural peptides targeting the scaffold activity of PI3Ky and their use in the treatment of pathologies of the respiratory apparatus .

[0005] BACKGROUND OF THE INVENTION

[0006] The 3' -5' -cyclic adenosine monophosphate (cAMP) second messenger controls different cellular functions, including cell growth and differentiation, gene transcription and protein expression. cAMP exerts its function through activation of different effectors, with protein kinase A (PKA) being the most widely characterized. cAMP directly binds the dimer of the regulatory subunits of the PKA holoenzyme, promoting the release of the two catalytic counterparts, which are then free to phosphorylate various substrates. Although different G protein-coupled receptors (GPCR) rely on the same second messenger cAMP for conveying signals within the cell, a tight spatial and temporal regulation of its concentration ensures that activation of a specific GPCR results in the appropriate cellular response (1) . This local control of cAMP signals is achieved through multiprotein complexes that sequester within subcellular microdomains enzymes responsible for cAMP generation (adenyl yl cyclases) and destruction (phosphodiesterases, PDEs) as well as distinct signal transducers. Key orchestrators of these "signaling islands" are A-kinase anchoring proteins (AKAPs) that, by definition, anchor PKA to its substrates and its regulators in specific cellular compartments ( 2 ) . Perturbations of this fine control of cAMP compartmentalization underlies different pathologies, including cardiovascular and pulmonary diseases, cancer, neurological disorders, and inflammation. On these grounds, pharmacological manipulation of specific cAMP signalosomes with molecules blocking the interaction of AKAPs with either PKA or other components of the cAMP signaling pathway has been attempted and proven effective in preclinical models (2, 3) .

[0007] Previous work from our group identified the AKAP phosphoinositide 3-kinase y (PI3Ky) as the orchestrator of a multiprotein complex which is central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways (4) . In the lungs, PI3Ky-bound PKA activates PDE4, ultimately restricting cAMP responses triggered by stimulation of p2-adrenergic receptors, the major GPCR mediating cAMP elevation in the airways. Displacement of the PI3Ky-anchored pool of PKA by a cell- permeable PI3Ky mimetic peptide (PI3Ky MP) inhibits PDE4 and promotes local cAMP elevation, eventually resulting in airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In bronchial epithelial cells, PI3Ky MP enhances cAMP in the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR) , the ion channel that controls mucus hydration, thereby driving the opening of the wild- type channel, while synergizing with CFTR modulators in reinstating the function of the most prevalent mutant in cystic fibrosis (F508del-CFTR) (4) . OBJECT AND SUMMARY OF THE INVENTION

[0008] An object of the present invention is to provide a potent PI3Ky / PKA disruptor for achieving therapeutic cAMP elevation in chronic respiratory disorders.

[0009] According to the invention, the above object is achieved thanks to the products specified in the ensuing claims, which are understood as forming an integral part of the present description.

[0010] The present invention concerns a non-naturally occurring peptide targeting PI3Ky with nanomolar affinity for PKA, high resistance to protease degradation and high permeability to the pulmonary mucus barrier able to disrupt the PI3Ky / PKA complex and to trigger cAMP elevation in the airways .

[0011] In one embodiment the present invention concerns a non-natural peptide having the ability of inhibiting the A-kinase anchoring function of PI3Ky comprising an amino acid sequence as set forth in SEQ ID No. : 1, wherein each amino acid is a D-amino acid, and wherein the glutamine at position 3 and the glycine at position 4 can be substituted with any amino acid with similar hydrophobicity, hydrophilicity, charge and size, respectively.

[0012] In further embodiments, the present invention concerns the non-natural peptide for use as a medicament, combination products of the non-natural peptide as well as pharmaceutical compositions comprising the same.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The invention will now be described, by way of example only, with reference to the enclosed figures of drawing, wherein :

[0015] - Figure 1. DRI-Pep#20 structure. a) Chemical structure of DRI-Pep #20. DRI-Pep#20 comprises the non-natural peptide RHQGK (SEQ ID No. : 1) , a glycine (G) linker and the retro-inverso sequence of the cell penetrating peptide penetratin 1 (Pl) , wherein each amino acid is a D-amino acid, b) Structural prediction of DRI-Pep#20 by PEP-FOLD3.5. The Pl-G domain is shown as cartoon in black. The key residues of the RHQGK domain, R- 1, H-2, Q-3 and K-5, are indicated and shown as sticks, c) Circular dichroism spectra of DRI-Pep#20 showing a peak at 190-240 nm. The percentage of oc-helical and -sheet secondary structures calculated by the K2D3 software are indicated. d) Molecular docking simulation of the interaction between DRI-Pep#20 and PKA by HADDOCK 2.4. The docked pose of DRI-Pep#20 in complex with residues 1-45 of PKA-RIIa is shown. The key residues involved in the binding are indicated (DRI-Pep#20 residues are in bold) and shown as sticks. Hydrogen bonds between DRI-Pep#20 and PKA-RIIa are indicated by dashed lines. The structural model was developed using PyMOL.

[0016] Figure 2. DRI-Pep#20 / PKA-RIIa (1-45) binding analysis by Optimal Hydrogen Bonding Network. a) Number of hydrogen bonds formed by R-l, H-2, Q-3, G-4, K-5 residues of DRI-Pep#20 with residues 1-45 of PKA- RIIa. b) Number of hydrogen bonds between residues 1-45 of PKA-RIIa with DRI-Pep #20. The interactions between PKA- RIIa and either Pl-G or RHQGK are shown in grey and black, respectively .

[0017] - Figure 3. DRI-Pep#20 binds PKA with high affinity. a) Schematic representation of the fluorescence spectroscopy assay for the characterization of the interaction between DRI-Pep#20 and the recombinant fluorescein 5-maleimide-labeled PKA-RIIa (PKA-F5M) . b) Steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep#20 (0 to 20 pM) . KD: dissociation constant. AU, arbitrary units. Inset, nonlinear fitting of the fluorescence intensity maxima obtained at various concentrations of DRI-Pep#20 for the monitoring of bio-labeled PKA. K : association constant, c) For kinetic analysis, fluorescence spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep#20 (0 to 5 pM) were analyzed and fitted to a single exponential function to obtain the observed rate constant ( ^s) . The binding of DRI-Pep#20 to bio-labeled PKA was investigated under pseudo- f irst-order conditions, and the kinetic constants, konand koffr were determined, d) Schematic representation of the displacement assay between DRI- Pep#20 and the PI3Ky / PKA-F5M complex. e) Percentage displacement of the PI3Ky / PKA-RIIa complex by DRI-Pep #20, calculated from steady-state emission spectra of the PI3Ky / PKA-F5M complex in the presence of increasing concentrations of DRI-Pep#20 (0 to 5 pM) . The displacement efficiency was expressed as percentage of the binding between PI3Ky and PKA-F5M in the absence of DRI-Pep #20. f) cAMP concentrations in peritoneal macrophages from wildtype (WT, in black) and PI3KY- / _mice (in grey) treated with 10-25 pM DRI-Pep#20 for 30 min. The amount of cAMP was expressed as percentage of cAMP accumulation observed in untreated PI3KY- / “ cells. n>6 technical replicates from N>3 independent experiments. ***P<0.001 WT versus PI3KY- / “ by one-way ANOVA, followed by Bonferroni's post hoc test. Data are means ± SEM.

[0018] - Figure 4. Potency and tolerability of DRI-Pep#20 in 16HBE14o- cells. a) cAMP elevation in 16HBE14o- cells in response to increasing concentrations of DRI-Pep#20 (31.6 nM to 316 mM range) for 30 min. The amount of cAMP was expressed as percentage of cAMP accumulation elicited by 100 mM DRI-Pep #20. n>9 technical replicates from N=3 independent experiments, b) Cell viability in 16HBE14o- cells treated with increasing concentration of DRI-Pep#20 (0 to 1000 pM) for 24 hours. N=3 independent experiments. In (a) and (b) , the median effective concentration (ECso) and the median lethal dose (LDso) were obtained by nonlinear regression analysis. LDso was calculated with respect to untreated control cells, whose viability was set to 100%. Throughout, data are means ± SEM.

[0019] - Figure 5. DRI-Pep#20 increases cAMP levels locally in vivo in the airway tract of mice. a) Schematic representation of the treatment schedule. BALB / c mice received DRI-Pep#20 through intratracheal (i.t. ) instillation, (b-d) cAMP concentrations in tracheas (b) , lungs (c) and hearts (d) from BALB / c mice 24 hours after i.t. instillation of different doses of DRI-Pep#20 (0 to 750 mg / kg) . Values in brackets indicate the dose of DRI- Pep#20 expressed as mg / kg. The number of mice (n) ranged from 3 to 6 per group. ECso, median effective concentration. In a) and b) , *P<0.05, **P<0.01, and ***P<0.001 by one-way ANOVA, followed by Bonferroni's post hoc test. Throughout, data are means ± SEM.

[0020] - Figure 6. DRI-Pep#20 can penetrate pathological mucus and resist protease degradation. a) Schematic representation of the Parallel Artificial Membrane Permeability Assay (PAMPA) with and without deposition of cystic fibrosis (CF) sputum on top of the artificial lipid membrane (PM) . b) Apparent permeability (Papp) measurements of DRI-Pep#20 (2 mg / mL) , in the absence and in the presence of CF sputum. The continuous line indicates the threshold Papp for high-medium permeable compounds (4xl0“6cm s-1) , while the dashed line defines the limit for medium-low permeable molecules (IxlO-6cm s-1) . ns: non-signif icant by Student' s t test, c) Representative Transmission Electron Microscopy (TEM) images of DRI-Pep#20 (0.1 mg / mL in water) . d) Size distribution profile of DRI-Pep#20 obtained by dynamic light scattering (DLS) analysis. e-f) cAMP concentrations in 16HBE14o- cells treated with DRI-Pep#20 (25 pM for 30 min) in the absence (black) and in the presence (grey) of either 3 pg / ml (e) or 20 pg / ml (f) human neutrophil elastase (HNE) . n>6 technical replicates from N>3 independent experiments. *P<0.05 and **P<0.01 by oneway ANOVA, followed by Bonferroni's post hoc test, ns: nonsignificant. g) cAMP elevation in 16HBE14o- cells covered with a layer of CF sputum and then treated with 25 pM DRI- Pep#20 for 30 min and 1 hour. The amount of cAMP was expressed as percentage of cAMP accumulation elicited by DRI-Pep#20 in the absence of sputum at 30 min. **P<0.01 and ***P<0.001 versus DRI-Pep#20 without sputum by two-way ANOVA test, followed by Bonferroni's post-hoc analysis. n>6 technical replicates from N>3 independent experiments. Throughout, data are means ± SEM.

[0021] - Figure 7. Prediction of protease cleavage sites within DRI-Pep #20. a) Prediction of protease cleavage sites within the DRI-Pep#20 sequence via Expasy PeptideCutter software, b) Prediction of protease cleavage sites within the DRI-Pep#20 sequence via Expasy PeptideCutter software and the Protease Specificity Prediction Server (PROSPER) . The cleavage sites within the Pl-G and RHQGK regions of DRI-Pep#20 are marked with an overbar and in bold, respectively. Figure 8. DRI-Pep#20 reinstates wild- type and F508del-CFTR activity, a) Schematic representation of CFTR activity measurement through the Premo™ Halide Sensor assay. b) Average fluorescence quenching traces of 16HBE14o- cells expressing the halide-sensitive yellow fluorescent protein (HS-YFP) and treated with either 25 pM DRI-Pep#20 or equimolar amount of the control peptide Pl for 30 min before addition of Premo Halide stimulus buffer. Fluorescence was continuously read (1 point per second) starting at 1 s before addition of the buffer and up to 120 s. The CFTR inhibitor CFTRinh-172 (10 pM for 5 min) was used to evaluate the selective activation of the CFTR channel, c) CFTR activity (expressed as the change in fluorescence AF / F0) in response to 30 min stimulation with increasing concentrations of DRI-Pep#20 (31.6 nM to 316 mM) in 16HBE14o- cells expressing HS-YFP. To determine the median effective concentration (ECso) , nonlinear regression analysis was used, d) CFTR activity (expressed as the change in fluorescence AF / F0) in 16HBE14o- cells expressing HS-YFP and treated with 10-25 pM DRI-Pep#20 for 30 min in the absence or in the presence of the CFTR inhibitor CFTRinh- 172 (10 pM for 5 min) . The adenylyl cyclase activator, forskolin (FSK) , was used as a positive control (100 nM for 5 min) , while Pl was used as a negative control (25 pM for 30 min) . UT : untreated cells, e) CFTR activity in F508del-CFTR-CFBE4 lo- cells expressing HS-YFP and treated with elexacaf tor / tezacaf tor / ivacaf tor alone (ETI) or together with DRI-Pep #20. Cells were corrected with elexacaftor (3 pM) and tezacaftor (10 pM) for 24 hours and then exposed acutely to ivacaftor (1 pM) for 30 min, alone (ETI) or together with 25 pM DRI-Pep #20. The CFTR inhibitor CFTRinh-172 was used as in (b) . UT : untreated cells. In (d) and (e) , n>3 technical replicates from N>3 independent experiments. **P<0.01, and ***P<0.001 versus UT and ### P<0.001 ETI versus ETI plus DRI-Pep#20 by oneway ANOVA, followed by Bonferroni's post hoc test. Throughout, data are means ± SEM.

[0022] - Figure 9. Binding of the RHQGK peptide to PKA. a) Schematic representation of the fluorescence spectroscopy assay for the characterization of the interaction between the RHQGK peptide and the recombinant fluorescein 5-maleimide-labeled PKA-RIIa (PKA-F5M) . b) Steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of the RHQGK peptide (0 to 1000 pM) . KD : dissociation constant. AU, arbitrary units. Inset, non-linear fitting of the fluorescence intensity maxima obtained at various concentrations of the RHQGK peptide for the monitoring of bio-labeled PKA. KA: association constant.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The invention will now be described in detail, by way of non-limiting examples.

[0025] In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments .

[0026] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0027] The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments .

[0028] In one embodiment, the present invention concerns a non-natural peptide having the ability of inhibiting the A-kinase anchoring function of PI3Ky comprising an amino acid sequence as set forth in SEQ ID No. : 1 (RHQGK) , wherein each amino acid is a D-amino acid, and wherein the glutamine at position 3 and the glycine at position 4 can be substituted with any amino acid with similar hydrophobicity, hydrophilicity, charge and size, respectively .

[0029] In one embodiment, the glutamine at position 3 can be substituted with asparagine, histidine and serine.

[0030] In one embodiment, the glycine at position 4 can be substituted with asparagine, serine and cysteine.

[0031] In one embodiment, the non-natural peptide further comprises a cell penetrating peptide.

[0032] In one embodiment, the cell penetrating peptide is selected from Penetratin (pAntp - RQIKIWFQNRRMKWKK - SEQ ID No. : 3) , HIV TAT peptide (YGRKKRRQRRR - SEQ ID No. : 4) , R7 peptide (RRRRRRR - SEQ ID No. : 5) , KALA peptide (WEAKLAKALAKALAKHLAKALAKALKACEA - SEQ ID No. : 6) , Buforin 2 (TRSSRAGLQFPVGRVHRLLRK - SEQ ID No. : 7) , MAP (KLALKLALKALKAALKLA-amide - SEQ ID No. : 8) , Transportan (GWTLNSAGYLLGKINLKALAALAKKIL-amide - SEQ ID No. : 9) , Transportan 10 (AGYLLGKINLKALAALAKKIL-amide - SEQ ID No. : 10) , Pvec ( LLI ILRRRIRKQAHAHSK-amide - SEQ ID No. : 11) , MPG peptide (GALFLGWLGAAGSTMGAPKKKRKV- amide - SEQ ID No. : 12) .

[0033] In one embodiment, the cell penetrating peptide sequence is retro-inversed with respect to the natural sequence. It is in fact commonly known that to preserve the biological function of a peptide synthesized using D- amino acids, the amino acid sequence can be presented in reverse (retro) order.

[0034] In one embodiment, the non-natural peptide further comprises a linker conjugating the amino acid sequence of SEQ ID No. : 1 with a cell penetrating peptide sequence.

[0035] In one embodiment, the linker is an amino acid linker.

[0036] In one embodiment, the linker comprises one or more glycines or one or more amino acids with hydrophobicity, hydrophilicity, charge, and size similar to glycine, e.g. , alanine, proline, serine.

[0037] In one embodiment, the linker is selected from the following sequences GG, GAG, GPA, GGGS (SEQ ID No. : 13) .

[0038] In one embodiment, the non-natural peptide comprises from N- to C-term the amino acid sequence of SEQ ID No. : 1, a linker, and a cell penetrating peptide.

[0039] In one embodiment, the linker and / or the cell penetrating peptides are made of D- amino acids.

[0040] In one embodiment, the cell penetrating peptide is made of D-amino acids and is retro-inversed compared to the natural sequence.

[0041] In one embodiment, the non-natural peptide has an amino acid sequence as set forth in SEQ ID No. : 2 (d (RHQGKGGKKWKMRRNQFWIKIQR) - in the following named DRI- Pep#20) . SEQ ID NO. : 2, which is entirely composed of D- amino acids, comprises the non-natural sequence SEQ ID No. : 1 (i.e., RHQGK) fused to the retro-inversed form of Penetratin 1 through a glycine (G) residue.

[0042] In one embodiment, the non-natural peptide, preferably the non-natural peptide of SEQ ID No. : 2, contains one or more modifications not altering the primary sequence, including chemical derivati zation, e.g. , acetylation, deuteration, Fmocylation.

[0043] In one embodiment, the present invention concerns the non-natural peptide as disclosed herein for use as a medicament .

[0044] In one embodiment, the present invention concerns the non-natural peptide as disclosed herein for use in treating respiratory diseases, preferably bronco-obstructive diseases .

[0045] In one embodiment, the respiratory diseases are selected from allergic asthma, cystic fibrosis (CF) , chronic obstructive pulmonary disease (COPD) , non-CF bronchiectasis, pulmonary hypertension (PH) , and idiopathic pulmonary fibrosis (IPF) .

[0046] In one embodiment, the non-natural peptide is suitable for administration by inhalation.

[0047] In one embodiment, the present invention concerns a product comprising: i) at least one non-natural peptide as disclosed herein, and ii) at least one potentiator of the cystic fibrosis transmembrane conductance regulator (CFTR) and / or at least one corrector of the cystic fibrosis transmembrane conductance regulator (CFTR) as a combined preparation for sequential, simultaneous or separate use in treating a respiratory disease. In one embodiment, the respiratory disease is a bronco-obstructive disease.

[0048] In one embodiment, the respiratory disease is cystic fibrosis .

[0049] In one embodiment, the potentiator of the cystic fibrosis transmembrane conductance regulator (CFTR) is selected from Ivacaftor or VX-770 (N- ( 2 , 4-Di- tert-butyl- 5 -hydroxyphenyl ) -1 , 4-dihydro-4-ossoquilonine-3- carboxamide ) , Navocaftor or ABBV-3067 ( [ 5- [ 3-amino-5- [ 4- ( trif luoromethoxy ) phenyl ] sul f onylpyridin-2-yl ] - 1 , 3 , 4- oxadiazol-2-yl] methanol ) and Deutivacaf tor or D9-ivacaftor (N- [ 2 -tert-butyl- 4 - [ 1 , 1 , 1 , 3 , 3 , 3-hexadeuterio-2- ( trideuteriomethyl ) propan-2-yl ] -5-hydroxyphenyl ] -4-oxo- lH-quinoline-3-carboxamide) .

[0050] In one embodiment, the corrector of the cystic fibrosis transmembrane conductance regulator (CFTR) is selected from Lumacaftor or VX-809 (3- (6-1 (2,2- dif lurobenzo [d] [1, 3] dioxol-5yl ) cyclopropanecarboxamido) - 3-methylpyridin-2-yl ) benzoic acid) , Tezacaftor or VX-661 (1- ( 2 , 2 -di fluoro- 1 , 3-benzodioxol- 5-yl ) -N- [l-[ ( 2R) -2 , 3- dihydroxypropyl ] -6-fluoro-2- (l-hydroxy-2-methylpropan-2- yl ) indol-5-yl ] cyclopropane-l-carboxamide ) , Elaxacaftor or VX- 445 (N- ( 1 , 3 -dime thylpyrazol-4 -yl ) sul f onyl- 6- [ 3- ( 3 , 3 , 3- tri fluoro- 2 , 2 -dimethylpropoxy) pyrazol-l-yl] -2- [ ( 4 S ) - 2,2, 4-trimethylpyrrolidin-l-yl ] pyridine- 3 -carboxamide ) , Galicaftor or ABBV-2222 ( 4 - [ ( 2R, 4R) - 4- [ [ 1- ( 2 , 2 -di f luoro-

[0051] 1, 3-benzodioxol- 5- yl ) cyclopropanecarbonyl ] amino] -7- ( di fluoromethoxy) - 3 , 4-dihydro-2H-chromen-2-yl ] benzoic acid) and Vanzacaftor ( ( 3Z, 14S ) -8- [ 3- ( 2- dispiro [ 2.0.24.13 ] hep tan- 7-yle thoxy ) pyrazol-l-yl ] - 12 , 12 - dimethyl-2 , 2 -di oxo -2X6 -thia- 3, 9, 11, 18,23- pentazatetracyclo[17.3.1.111,14.05, 10] tetracosa- l (22) ,3, 5 (10) , 6,8, 19 (23) , 20-heptaen-4-olate ) .

[0052] In one embodiment, the present invention concerns a pharmaceutical composition comprising at least one nonnatural peptide as disclosed herein and a pharmaceutically acceptable vehicle.

[0053] In one embodiment, the pharmaceutically acceptable vehicle is selected among phosphate buffered saline, saline, hypertonic saline, water.

[0054] In one embodiment, the pharmaceutical composition comprises at least one excipient.

[0055] In one embodiment, the pharmaceutical composition comprises at least one excipient selected among NaCl, HC1, NaOH, EDTA, Polysorbate 20, Polysorbate 80, Citric acid, Na citrate, K phosphate, Na phosphate, Na bicarbonate, Ascorbic acid, Lactose, Glucose, Mannitol, Trehalose, Sucrose, Mg stearate, Glutathione, Vitamin E, Cyclodextrin, DPPC, DSPC, DMPC, Leucine, Isoleucine, Methionine, Histidine, Glycine, Poloxamer, Chitosan, Trimethylchitosan, PLGA, PEG.

[0056] The unexpectedly high resistance to degradation and potency of DRI-Pep#20 renders this compound the ideal candidate for therapeutic cAMP modulation, especially for the treatment of chronic respiratory diseases, being the PI3Ky-PKA signaling hub central to multiple functions of airway cells, like smooth muscle relaxation, epithelial ion transport and neutrophil inf il tration ( 4 ) . Our observations that DRI-Pep#20 can be efficiently delivered locally in the airways, and that its biological activity is completely preserved in the presence of human neutrophil elastase, support the possibility of using this peptide to ensure therapeutically relevant cAMP elevation in highly inflamed lungs. This is relevant for patients with COPD, non-CF bronchiectasis and certain forms of asthma, but also the lethal genetic disease CF, since airway inflammation is not eliminated in these patients, despite the introduction of highly effective modulator therapies (HEMT) targeting the basic genetic defect of the disease (5, 6) .

[0057] In addition to inflammation-related proteases, another barrier imposed by diseased lungs that may hamper the bioavailability of inhaled therapeutics is the thick mucus layer covering the respiratory epi thelia ( 7 ) , especially in CF individuals, as well as in patients with COPD, non-CF bronchiectasis and asthma. Our cell-based assays using patient-derived sputum as a proxy of CF mucus reveal a good mucus permeability of DRI-Pep #20, which likely stems from its molecular dimensions being compatible with that of the meshes of the pathological mucus. TEM and DLS analysis indicate that the peptide can form aggregates of 200 nm in size, which could freely diffuse through the 100-1000 nm meshes of the network of bundled fibers that are typically formed by biopolymers of the CF mucus and that are filled with a low viscosity fluid(8) .

[0058] In virtue of its ability to efficiently penetrate mucus layers, DRI-Pep#20 can be exploited to achieve therapeutic cAMP elevation in CF bronchial epithelial cells, specifically in PI3Ky-directed subcellular compartments, which we previously show to positively affect CFTR activity and maximize the effect of HEMT (4) . This is supported by our data showing that DRI-Pep#20 doubles the effects of the gold-standard combination of CFTR modulators, elexacaf tor / tezacaf tor / ivacaf tor in rescuing the activity of the most common CFTR mutant, F508del. These findings have important clinical implications in light of recent studies showing that CFTR potentiators and correctors restore only partially the function of mutant channels, up to 60% of the levels of the wild-type CFTR(9- 11) , with consequent residual disease in CF patients treated with HEMT (5, 6, 12) . Thus, DRI-Pep#20 offers the possibility of significantly increasing the efficacy of the standard of care for CF patients.

[0059] In conclusion, DRI-Pep#20 can be used for achieving therapeutic cAMP elevation locally in the lungs, in chronic respiratory disorders with high unmet medical need, such as the lethal genetic disease CF.

[0060] Results

[0061] DRI-Pep#20 structure and biological properties.

[0062] The chemical structure of DRI-Pep#20 is shown in Fig. la (d (RHQGKGGKKWKMRRNQFWIKIQR) - SEQ ID No. : 2) . Predictions of the tridimensional structure of the peptide suggested the presence of an a-helix, flanked by two uncoiled regions (Fig. lb) . The presence of a-helix structures (18.51%) was confirmed by circular dichroism analyses showing a double-peak signal, with a maximum at 200 nm which is typical for a-helix structures, and a minimum in the 220-240 nm region which is characteristic for random-coil regions (13) (Fig. 1c) . In silico simulations of the binding of DRI-Pep#20 to amino acids 1- 45 of PKA-RIIa, the typical binding surface for AKAPs, revealed that the kinase could form hydrogen bonds with the RHQGK sequence, mainly involving the arginine and the lysine in position 1 and 5, respectively, and to a lesser extent histidine 2 and glutamine 3 (Fig. Id, Fig. 2 and Table 1) . Table 1. Binding energetics and kinetics of DRI-Pep#20 obtained through molecular docking analysis.

[0063] The high affinity of the DRI-Pep#20 for PKA was further corroborated by in vitro experiments showing that the peptide associated recombinant PKA-RIIa with a dissociation constant (KD) in the nanomolar range (76 nM; Fig. 3a-b) . Further fluorescence spectroscopy experiments confirmed that the RHQGK sequence of DRI-Pep #20 was able to directly bind PKA (Fig. 9a-b) . In addition, stoppedflow fluorescence assays revealed that the binding of DRI- Pep#20 to PKA-RIIa was extremely fast, with a Konof 10~6s, while the dissociation process was significantly slow, being the KOff 10~2s (Fig. 3c and Table 2) .

[0064] Table 2. Binding kinetics of the interaction between

[0065] In agreement with the high affinity of DRI-Pep#20 to PKA-RIIa, the peptide displaced the binding between recombinant PI3Ky and PKA-RIIa with an efficiency as high as 74% (Fig. 3d-e) . Notably, despite the elevated binding affinity to PKA-RIIa, DRI-Pep#20 retained the selectivity for the PI3Ky-bound pool of PKA since the peptide failed to increase cAMP in cells devoid of PI3Ky (Fig. 3f) .

[0066] Dose-response experiments showed that DRI-Pep#20 increased cAMP levels in 16HBE14o- cells with an ECso of 15 pM (Fig. 4a) and it was well tolerated, being the LD50 10- fold higher than the ECso (Fig. 4b) .

[0067] Overall, these results identify DRI-Pep#20 as a selective PI 3Ky / PKA-RIIa peptide disruptor with high affinity to PKA-RIIa.

[0068] DRI-Pep#20 has good mucus permeability and protease resistance

[0069] Next, we sought to determine to what extent DRI-Pep#20 could be used for modulating cAMP in the lungs for therapeutic purposes. First, we assessed the suitability for local delivery to the airways. Following intratracheal instillation (Fig. 5a) , DRI-Pep#20 induced a dosedependent increase in cAMP levels in the trachea and in the lungs, with an EC50 of 8.06 pg / Kg and 11.78 pg / Kg, respectively (Fig. 5b-c) . Of note, cardiac cAMP metabolism was unchanged (Fig. 5d) , suggesting that the peptide increased cAMP locally in the lungs without systemic effects .

[0070] Because the efficacy of inhaled therapies can be hampered by extracellular barriers imposed by diseased lungs, such as a thick layer of protease-rich mucus (7) , we next sought to determine to what extent DRI-Pep#20 could penetrate mucus layers and resist to protease degradation. DRI-Pep#20 penetrated the phospholipid membrane of the PAMPA system (Fig. 6a) with an apparent permeability (Papp) of 1.88 x 10~6cm s-1(Fig. 6b) . Of note, the addition of pathological OF sputum on top of the phospholipid layer (Fig. 6a) did not significantly affect the Pappof the peptide (Papp2.55 x 10~6cm s-1) (Fig. 6b) . To verify whether the good mucus permeability of DRI-Pep#20 could be ascribed to molecular dimensions compatible with the mesh size of CF mucus (14) , Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) assays were performed. TEM images show that DRI-Pep#20 formed irregular aggregates of 5-40 nm in size (Fig. 6c) , which was in agreement with the particle diameter of 10-20 nm retrieved by DLS analysis (Fig. 6d) .

[0071] Next, we tested whether DRI-Pep#20 retained the ability to elevate cAMP in pulmonary cells in the presence of neutrophil elastase, the most abundant protease in the lungs of patients with neutrophilic airway diseases, like COPD and CF(15) . The ability of the peptide to raise cAMP in 16HBE41o- cells was completely unaltered by the presence of 3 pg / ml of recombinant human neutrophil elastase (HNE) (Fig. 6e) , a dose which was previously shown to inactivate other therapeutic peptides (16) . Notably, the activity of the DRI-Pep#20 was completely preserved even in the presence of a 10-fold higher concentration of HNE (Fig. 6f ) , an amount that is typically detected in the lungs of patients with severe bronchiectasis ( 15) , which was in agreement with the absence of any predicted cleavage sites by HNE (Fig. 7a) . The good resistance of DRI-Pep#20 to degradation was confirmed in the presence of a more complex biological matrix containing other proteases that could potentially cleave the peptide (Fig. 7b) , that is CF sputum, where the DRI-Pep#20 retained 72% of its biological activity (Fig. 6g) .

[0072] Taken together, these data demonstrate the ability of DRI-Pep#20 to elevate lung cell cAMP in the presence of a hostile extracellular environment composed of a mucus barrier enriched in proteases, which is typical of diseased lungs . DRI-Pep#20 promotes cAMP-dependent activation of wild-type and F508del-CFTR in human bronchial epithelial cells

[0073] Next, we aimed to assess the extent to which DRI- Pep#20 could effectively restore cAMP levels and consequently reactivate the activity of CFTR, a cAMP- dependent chloride channel impaired in a range of respiratory disease, primarily including CF(17) . First, we assessed the ability of the peptide to stimulate the activity of the wild-type channel in 16HBE141o- cells expressing the halide-sensitive yellow fluorescent protein (HS-YFP) (Fig. 8b) , which allows quantifying CFTR activity based on the fluorescence quenching rate elicited by an iodide influx (18) . DRI-Pep#20 induced a 60% reduction in YFP fluorescence, which was completely prevented by coapplication of the CFTR inhibitor, CFTRinh-172 (Fig. 8b) , demonstrating selective activation of CFTR channels. Doseresponse experiments revealed an ECso of 20 pM (Fig. 8c) and demonstrated that 25 pM DRI-Pep#20 was as effective as 10 pM forskolin, the adenylyl cyclase activator, in triggering CFTR gating in 16HBE141o- cells (Fig. 8d) .

[0074] Next, we assessed to what extent DRI-Pep#20 could reinstate the activity of F508del-CFTR in combination with the standard of care for CF, including two CFTR correctors (Elexacaftor / Tezacaf tor ) and one CFTR potentiator (Ivacaftor) , that partially rescue the trafficking and gating defects of the mutant channel, respectively ( 19 ) . In CF bronchial epithelial cells overexpressing the F508del- CFTR mutant and the HS-YFP, Elexacaf tor / Tezacaftor / Ivacaftor (ETI) reduced YFP fluorescence of 50%, which was further decreased down to 25% when DRI-Pep#20 was added together with ETI (Fig. 8e) .

[0075] Hence, these data support the use of DRI-Pep#20 as a single agent or as an-add on to CFTR modulators, to therapeutically stimulate the activity of wild-type and F508del CFTR, respectively.

[0076] Material and Methods

[0077] Peptides and reagents

[0078] Peptides were synthesized by GenScript (Piscataway, NJ) at >95% purity.

[0079] Recombinant human PKA regulatory subunit Rlla (PKA- Rlla) and PI3Ky catalytic subunit (pllOy) were purchased by Biaffin GmbH & Co KG (product code: PK-PKA-R2A025 , Kassel, DE) and Origene Technologies (TP307790, Rockville, US) , respectively .

[0080] Human neutrophil elastase was purchased from Sigma- Aldrich (CAS 9004-06-2, Sigma-Aldrich, Saint Louis, MO) and reconstituted in 50 mN sodium acetate, pH 5.5, with 200 mN NaCl . VX-809 ( Lumacaf tor ) , VX-770 (Ivacaftor) , VX- 661 (Tezacaftor) and VX-445 (Elexacaf tor) were purchased from MedChemExpress LLC (Princeton, USA) . Forskolin and CFTRinh -172 were purchased from Sigma -Aldrich (CAS 66575- 29-9, Sigma-Aldrich, Saint Louis, MO) .

[0081] Cell lines

[0082] Immortalized human bronchial epithelial cells expressing wild-type CFTR (16HBE14o-) or F508del-CFTR (CFBE41O-) were purchased from Sigma-Aldrich (16HBE14o-, product code: CAS SCC150, CFBE41O-, product code: CAS SCC151, Sigma -Aldrich, Saint Louis, MO) . Cells were grown in Minimum Essential Medium (MEM) supplemented with 10% FBS, 5 mM L-Glutamine, 100 U / ml penicillin and 100 pg / ml streptomycin (Thermo Fisher Scientific, Waltham, MA) on culture dishes pre-coated with human fibronectin (1 mg / ml; Sigma -Aldrich, Saint Louis, MO) , bovine collagen I (3 mg / ml; Sigma- Aldrich, Saint Louis, MO) and bovine serum albumin (0.1%; Sigma -Aldrich, Saint Louis, MO) diluted in LHC-8 basal medium (Invitrogen, Waltham, MA) . Cells up to passage 15 were used for experiments. All cells were cultured at 37°C and under a 5% CO2atmosphere.

[0083] Animals

[0084] PI3Ky_def icient mice (PI3KY- / “) were described previously (4) . Mutant mice were back-crossed with C57Bl / 6j mice for 15 generations to inbreed the genetic background and C57Bl / 6j were used as controls (WT) . Mice used in all experiments were 8 to 12 weeks of age. Mice were group- housed, provided free-access to standard chow and water in a controlled facility providing a 12-hour light / dark cycle and were used according to institutional animal welfare guidelines and legislation, approved by the local Animal Ethics Committee. All animal experiments were approved by the animal ethical committee of the University of Torino and by the Italian Ministry of Health (Authorization n° 757 / 2016-PR) and the obligations of Legislative Decrees No. 206 of April 12, 2001, and No. 224 of July 8, 2003 have been met.

[0085] Isolation of murine peritoneal macrophages

[0086] Peritoneal macrophages were prepared from 8- to 12- week- old wild-type (WT) and PI3KY- / “ mice, as described previously (4) . Briefly, cells were collected from euthanized animals by peritoneal lavage with 5 mL of PBS, supplemented with 5 mM EDTA. Cells were centrifuged for 3 min at 300 g and the pellet was resuspended in culture media including Roswell Park Memorial Institute (RPMI) media, 100 U / ml penicillin and 100 pg / ml streptomycin, and 10% heat-inactivated FBS (Thermo Fisher Scientific, Waltham, MA) . Macrophages were seeded in 96-well plates (l*106cells / well) and maintained at 37°C with 5% CO2 for at least 16 / 18 h before treatment with the peptide and cAMP quantification. cAMP measurements

[0087] From cells: cAMP content was measured in 16HBE14o- cells at the indicated time points after treatment with the indicated doses of peptides using the Promega cAMP- Glo™ Assay kit (Promega, Milano, IT) , according to the manufacturer' s protocol.

[0088] From tissues: lungs, tracheas and hearts were collected from euthanized mice 24 h after intratracheal instillation of different doses of the peptide (0 to 750 mg / kg in a final volume of 50 pl of PBS) . Snap-frozen tissues were powdered in liquid nitrogen and extracted with cold 6% trichloroacetic acid. Samples were sonicated for 10 sec, incubated at 4 °C under gentle agitation for 10 min and then centrifuged at 13000 rpm at 4 °C for 10 min. Supernatants were washed four times with five volumes of water saturated with diethyl ether and lyophilized. cAMP content was detected with Cyclic AMP ELISA Kit (Cayman Chemical, Michigan, USA) , according to the manufacturer's protocol .

[0089] Cell viability assay

[0090] Human bronchial epithelial cells (16HBE14o-) were seeded in 96-well plates (2*104cells / well) and incubated for at least 16 / 18 hours at 37°C with 5% CO2 before experiments. Non-adherent cells were eliminated by washing with PBS and cells were then stimulated with 8 different doses of the indicated peptide (0 pM - 1 mM range) for 24h. ATP levels were evaluated as an indicator of viable cells using the Cell Titer-Gio® Luminescent Cell Viability Assay (Promega, Milano, IT) , according to the manufacturer's protocol. The lethal dose (LD50) was calculated with respect to untreated control cells, whose viability was set to 100%.

[0091] CFTR activity measurements

[0092] CFTR-mediated anion transport was measured by using the Premo™ Halide Sensor (Thermo Fisher Scientific, Waltham, MA) which allows assessing CFTR activity by measuring the rate of YFP fluorescence quenching caused by iodide / chloride exchange across the plasma membrane. Briefly, the halide-sensitive yellow fluorescent protein (HS-YFP) was expressed in 16HBE14o- and F508del-CFTR- CFBE41o- cells through the BacMam technology, according to the manufacturer's protocol. Cells expressing the HS-YFP were cultured on 96-well plates and treated with the indicated peptides / compounds for the indicated time. Fluorescence was evaluated in a plate reader immediately after addition of 150 pl of Halide stimulus buffer (an Nal- containing solution) leading to a final Nal concentration in the wells of 75 mM. Fluorescence was continuously read (1 point per second) starting at 1 s before Halide stimulus buffer addition and up to 120 s. CFTR activity was expressed as AF / Fo where AF was obtained by subtracting the background fluorescence (fluorescence of cells not expressing HS-YFP) to the fluorescence measured at the specific time point after addition of Nal . AF was then normalized to the initial fluorescence Fo (fluorescence of HS- YFP-expres sing cells immediately after addition of Nal) to obtain a measure of relative fluorescence AF / Fo.

[0093] CF sputum samples

[0094] Spontaneous expectorated sputum samples from CF patients in stable clinical conditions were collected at the Bronchiectasis and Cystic Fibrosis Programs of the Respiratory Department of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico in Milan (Italy) and processed as previously described ( 20 ) . The patients signed an express, free and informed, consent to the collection and use of their biological samples. Briefly, samples were processed getting first rid of saliva, sputum plugs were then selected and weighted. Samples were diluted 8X in PBS, vortexed until sputum dissolution and centrifuged for 15min at 3000 g. Supernatants were recovered and stored at -80 °C, thawed overnight at 4 °C, and all subsequent experiments were undertaken within 24h from thawing. Neutrophil elastase was quantified as described previously (20 ) and sputum samples containing 20 pg / mL of active neutrophil elastase were used to assess the activity of peptides in 16HBE14o- cells in the presence of CF sputum. Briefly, cells were seeded in 96-well plates (2*104cells / well) and incubated for at least 16 / 18 h at 37°C with 5% CO2 before experiments. Subsequently, peptides were diluted in PBS at a final concentration of 25 pM and a PBS: sputum mixture (1:1) was added on the top of adherent cells (100 pl / well) and cAMP levels were quantified at the indicated time points using the Promega cAMP-Glo™ Assay kit (Promega, Milano, IT) , according to the manufacturer's protocol .

[0095] PAMPA assay

[0096] To assess the permeability of peptides through a CF sputum layer, a parallel artificial membrane permeability system (PAMPA) (Corning Gentest Pre-coated PAMPA, 353015, USA plates) that allows to measure the ability of drugs to diffuse from a donor compartment, through an artificial membrane, into an acceptor compartment, was used as described previously ( 21 ) . The bottom wells of the PAMPA system ("acceptor" wells) were filled with 300 pL of PBS (lOmM, 150 mM NaCl, pH 7.4) , while "donor" wells were filled with 200 pL of the peptide solution (2 mg / mL in lOmM PBS, 150 mM NaCl, pH 7.4) , in the absence or in the presence of CF sputum. In the latter case, 40 pL of CF sputum was first deposited over the PAMPA membrane, and the peptide solution was subsequently added over the CF sputum layer. Afterwards, the two wells were coupled and incubated for 5 h at RT . At the end of the incubation, the plates were splitted and the amount of peptide diffused into the acceptor well was quantified by fluorescence spectroscopy using a Horiba Jobin Yvon Fluorolog 3 TCSPC fluorimeter (Horiba, Kyoto, Japan) equipped with a 450-W xenon lamp and a R928 photomultiplier (Hamamatsu Photonics, Hamamatsu, Japan) . Excitation was performed at 280nm while emission was recorded the wavelength region 290-500 nm (maximum of emission at 362 nm) . Excitation and emission slits were set at 4 and 5 nm, respectively. The concentration of the peptide was calculated using a 6- points calibration curve. The apparent permeability coefficient (Papp) was expressed according to this relationship : derived from Fick's law for steady-state condi tions ( 22 ) , where dQ is the quantity of peptide expressed as moles permeated into the acceptor compartment at time t (18000 sec) , Co is the initial concentration of the peptide in the donor well, and A is the area of the well membrane (0.3 cm2) .

[0097] PKA-RIIa bioconjugation and fluorescence spectroscopy

[0098] Recombinant PKA-RIIa was bio-conjugated to fluorescein 5-maleimide (F5M) , as described previously (23) using 75 pg of PKA-RIIa and a 50-fold excess of F5M. After biocon ugation, the derivative was immediately purified using a Sephadex® G-25 desalting column and phosphate-buf f ered saline solution (PBS) (20 mM, 150 mM NaCl, pH 7.2) as eluent. To evaluate F5M labelling efficiency, the dye / protein ratio (D / P) of the conjugates was determined by the absorption spectra of the labelled proteins in PBS (20 mM, 150 mM NaCl, pH 7.2) , according to the following equation: where A280 is the absorption of the conjugate at 280 nm; Amax is the absorption of the conjugate at the absorption maximum of the corresponding F5M; c is a correction factor (which must be used to normalize the A280 signal because fluorescent dyes (i.e. F5M) also absorb at 280 nm and equals the A280 of the dye divided by the Amax of the dye (c = 0.29) ; eProt (25, 169 M-1cm_1) and edye (63,096 M-1cm-1) are the molar extinction coefficients of PKA and F5M, respectively. PKA-RIIa presents six cysteine residues, and the final D / P value was 0.2. UV-visible absorption spectra were measured with a UH5300 spectrophotometer (Hitachi, Tokyo, Japan) at RT, using 1 cm pathway length quartz cuvette. Fluorescence emission spectra in steady-state mode were acquired at RT using a Jobin Yvon Fluorolog 3 TCSPC fluorimeter (Horiba, Kyoto, Japan) equipped with a 450-W Xenon lamp and a R928 photomultiplier (Hamamatsu Photonics, Hamamatsu, Japan) . Steady-state fluorescence spectra were recorded in the 500- 600 nm range. The excitation 2 wavelength was set on 490 nm and the excitation and emission slits were set on 2 and 4, respectively. Equilibrium binding constants (KD and K ) were obtained from steady-state data.

[0099] Fluorescence kinetics were measured using an Applied Photophysics SX20 stopped-flow spectrophotometer (Applied Photophysics, North Carolina, US) fitted with a 495 nm cutoff filter between the cell and the fluorescence detector and equipped with a thermostat bath set at 2510.2 °C. Association and dissociation rate constants ( konand kOff) were calculated from stopped-flow kinetics data. Data acquisition, visualization and analysis were performed with Pro-Data software from Applied Photophysics Ltd (Applied Photophysics, North Carolina, US) .

[0100] To assess the ability of DRI-Pep#20 to displace the binding between PI3Ky and PKA-F5M, steady-state emission spectra of the PI 3Ky / PKA-F5M complex in the presence of increasing concentrations of the peptide were acquired. Briefly, 50 nM of recombinant PI3Ky was added to 100 nM F5M-bounded PKA-RIIoc in a total volume of 100 pL PBS. The concentration of the PI 3Ky / PKA-F5M complex was kept constant while gradually titrated with increasing concentrations of the peptide from 0 to 5 pM. The complex was excited at 490 nm and emission spectra were recorded in the 500-600 nm spectral range, as described above. The degree of displacement of the PKA-RIIoc-PI3Ky complex was expressed as the percentage of fluorescence quenching after addition of the peptide.

[0101] Circular dichroism

[0102] Circular dichroism (CD) measurements were performed on a Jasco-810 Dichrograph equipped with a Peltier thermoelectric controller (Jasco Inc. , Easton, US) . The spectra of peptides were recorded in the continuous mode between 260 and 180 nm at 25 °C in 0.1 cm path length quartz cuvette (Hellma GmbH, Mullheim, DE) with a total peptide concentration of 0.2 mg / mL dissolved in 2 mM PBS (0.6 mM KH2PO4, 1. 6 mM K2HPO4) , pH 7.4. The CD spectrum in the 190-240 nm range was used to predict the secondary structural content of the peptide using the K2D3 web server (24) .

[0103] Transmission Electron Microscopy (TEM) and Dynamic

[0104] PKA-RIIa structure prediction

[0105] The 3D structure of residues 1-45 of PKA-RIIa (SEQ ID No. : 14 - MSHIQI PPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA) was predicted using the Iterative Threading ASSEmbly Refinement (I-TASSER) web server (25) , an on-line platform that implements I-TASSER-based algorithms for predictions of protein structure and function. Briefly, starting from the FASTA amino acid sequence I-TASSER ran a three steps simulation, first threading it through a representative PDB structure library to search for possible template folds or supersecondary-structure fragments, using a profileprofile alignment-based threading algorithm. In the second step, the continuous fragments excised from the PDB templates were reassembled into full-length models, while the unaligned regions were built by ab initio modeling. Finally, the structure trajectories were clustered, and the lowes t-energy structures selected, and an all-atom model was constructed by REMO41 through optimization of the hydrogen-bonding network. The five best models obtained by I-TASSER were subsequently evaluated based on their threading template and predicted C-score. The model with the highest C-score of -0.22 and predicted using the NMR structure of PKA-RIIa as a threading template (PDB ID 2KYG) (26) was selected.

[0106] DRI-Pep#20 Structure Prediction

[0107] The structure of DRI-Pep#20 was predicted with PEP- FOLD3.5 (27) , a de novo approach that predicts peptide structures from amino acid sequences. Briefly, starting from the amino acid sequence, first a series of 200 simulations was run, each one sampling a different region of the conformational space using the Generator taboosampling 5 (ts5) , recommended for peptides longer than 10 amino acids . The output was an archive of clusters of all the models sorted out using the TM score followed by performing the Model Quality assessment using Apollo (28) . The first five models, representing the five best conformation of each cluster with the best scores defined according to the lowest sOPEP energy and the highest TM- score value, were selected and further supported by RMSD. Finally, the best structure of the peptide was validated by visual analysis on PYMOL .

[0108] PKA-RIIa-DRI-Pep#20 docking

[0109] PKA-RI la-DRI -Pep#20 docking studies were performed with the High Ambiguity Driven Biomolecular DOCKing (HADDOCK) software. Within the HADDOCK process, the residues 1-45 of PKA-RIIa were selected as the active residues and enforced to be part of the interface by applying ambiguous interaction restraints. Briefly, starting from the PKA-RIIa and the DRI-Pep#20 structure, the HADDOCK docking ran three consecutive steps, first the molecules were randomly oriented, and a rigid-body search was performed (itO) . The output was an archive of 1000 models, among them the top 200 ranked structures were selected based on the energy function and addressed to the semi- flexible simulated annealing stage performed in torsion angle space (itl) . In the third stage, the structures were refined in Cartesian space with explicit solvent layer (water) and subjected to a short molecular dynamic simulation at 300K. During the refinement, both the side chain and backbone of interface residues were progressively allowed to move.

[0110] The final models were automatically clustered based on the positional interface ligand RMSD (iL-RMSD) by fitting the conformational changes on the interface of the receptor (PKA-RIIa) and on the interface of the smaller partner (the peptides) . Finally, the protein-peptide binding poses were assessed by the HADDOCK report and the binding affinity was evaluated by the Optimal Hydrogen Bonding Network. The resulting best binding pose was validated by visual analysis on PYMOL .

[0111] Statistical analysis

[0112] Data are presented as scatter plots with bars (means ± SEM) . Prism software (GraphPad Software Inc. ) was used for statistical analysis. Raw data were first analyzed to confirm their normal distribution via the Shapiro-Wilk test and then analyzed by unpaired Student's t test, one-way analysis of variance (ANOVA) , or two-way ANOVA. Bonferroni correction (one-way and two-way ANOVA) was applied to correct for multiple comparisons. P < 0.05 was considered significant .

[0113] References

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Claims

CLAIMS1. A non-natural peptide having the ability of inhibiting the A-kinase anchoring function of PI3Ky comprising an amino acid sequence as set forth in SEQ ID No. : 1, wherein each amino acid is a D-amino acid, and wherein the glutamine at position 3 and the glycine at position 4 can be substituted with any amino acid with similar hydrophobicity, hydrophilicity, charge and size, respectively .

2. The non-natural peptide according to claim 1, wherein the peptide further comprises a cell penetrating peptide .

3. The non-natural peptide according to claim 2, wherein the cell penetrating peptide is selected from Penetratin (pAntp) , HIV TAT peptide, R7 peptide, KALA peptide, Buforin 2, MAP, Transportan, Transportan 10, pVEC, MPG peptide.

4. The non-natural peptide according to claim 2 or claim 3, wherein the peptide further comprises a linker conjugating the amino acid sequence of SEQ ID No. : 1 with the cell penetrating peptide.

5. The non-natural peptide according to any one of the preceding claims, wherein the cell penetrating peptide sequence is retro-inversed with respect to the natural sequence .

6. The non-natural peptide according to claim 4 orclaim 5, wherein the linker is an amino acid linker comprising one or more glycine amino acids and / or one or more amino acids with hydrophobicity, hydrophilicity, charge, and size similar to glycine.

7. The non-natural peptide according to any one of the preceding claims having an amino acid sequence as set forth in SEQ ID No . : 2.

8. The non-natural peptide according to any one of the preceding claims for use as a medicament.

9. The non-natural peptide according to any one of the preceding claims for use in treating respiratory diseases, preferably broncho-obstructive diseases.

10. The non-natural peptide for use according to claim 9, wherein the respiratory diseases are selected from allergic asthma, cystic fibrosis, chronic obstructive pulmonary disease, non-cystic fibrosis bronchiectasis, pulmonary hypertension, and idiopathic pulmonary fibrosis.

11. The non-natural peptide for use according to any one of claims 8 to 10, wherein the non-natural peptide is suitable for administration by inhalation.

12. A product comprising: i) at least one non-natural peptide according to any one of claims 1 to 7 , and ii) at least one potentiator of the cystic fibrosis transmembrane conductance regulator (CFTR) and / or at least one corrector of the cystic fibrosis transmembraneconductance regulator (CFTR) as a combined preparation for sequential, simultaneous or separate use in treating respiratory diseases.

13. Product according to claim 12, wherein the potentiator of the cystic fibrosis transmembrane conductance regulator (CFTR) is selected from Ivacaftor, Navocaftor and Deuti vacaf tor .

14. Product according to claim 12 or claim 13, wherein the corrector of the cystic fibrosis transmembrane conductance regulator (CFTR) is selected from Lumacaftor, Tezacaftor, Elaxacaftor, Galicaftor and Vanzacaftor.

15. A pharmaceutical composition comprising at least one non-natural peptide according to any one of claims 1 to 7 and a pharmaceutically acceptable vehicle.

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